Protection against lung infection by maternal immunization with PspA among offspring.

<p>Seven-day-old mice were intranasally challenged with 5×10⁵ CFU TIGR4 strain (10 µl/mouse) with anesthesia. Three days after challenge, lungs were collected and the numbers of pneumococci colonies in the lung homogenate were determined. Each dot shows the Log₁₀ CFU/mouse. Each horizontal line shows the median Log₁₀ CFU/mouse. Group A (n = 18), B (n = 16), C (n = 20), and D (n = 27) mice were produced in the same manner as the corresponding groups in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027102#pone-0027102-g003" target="_blank">figure 3</a>. <i>p</i><0.05 and <i>p</i><0.01 are <i>p</i>-values for differences between the indicated group and the non-immune mice in Group D by Kruskal-Wallis test with Dunn's multiple comparison test.</p

Anti-PspA specific IgG subclasses in sera and milk of mother mice.

<p>Female mice were intranasally immunized twice each week with 1 µg of rPspA and 4 µg CTB for first 2 weeks and with 1 µg rPspA alone for the last week. The levels of anti-PspA specific IgG1, IgG2a, IgG2b and IgG3 antibodies in sera (A) and breast milk (B) were determined by PspA-specific ELISA on day 0, 7 and 14 after the birth. The values shown are the mean ± S.E. concentrations (ng/ml) taken from PspA-immunized mother (n = 16) and sham-immunized mother (n = 14). The mean values of IgG1/IgG2a antibody to PspA in the sera of the individual mother's sera were 3.1, 2.1 and 1.1 for day 0, 7, and 14, respectively. The mean IgG1/IgG2a anti-PspA values for the individual mother's milk samples were 5.7, 6.5, and 1.7 for day 0, 7, and 14, respectively. The levels of anti-PspA specific IgG subclasses in sera and breast milk from sham-immunized mice were below the detections limit. * <i>p</i><0.05 when compared with mice at day 0 by ANOVA test or Kruskal-Wallis test with Dunn's multiple comparison test. n.d. not determined.</p

Protection against nasal carriage of pneumococci by maternal immunization with PspA among offspring.

<p>Offspring at 7-day-old were intranasally challenged with 1×10⁵ CFU TIGR4 strain (5 µl/mouse) without anesthesia. Two days after challenge, nasal washes and homogenized washed nasal tissues were collected and the numbers of pneumococci colonies were determined. No evidence of protection was observed in CFUs in nasal washes (not shown). Results are shown for CFU in homogenized washed nasal tissue. Each dot represents the Log₁₀ CFU/mouse. Each horizontal line depicts the median Log₁₀ CFU/mouse. Group A (n = 11), B (n = 10), C (n = 13), and D (n = 15) mice were produced in the same manner as the corresponding groups in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027102#pone-0027102-g003" target="_blank">figure 3</a>. Group A differed from Group D at <i>p</i><0.05 by Kruskal-Wallis test with Dunn's multiple comparison test.</p

Anti-PspA specific IgG subclasses in sera of offspring.

<p>The levels of anti-PspA specific IgG subclasses in offspring's sera were determined by PspA-specific ELISA on day 0, 7, and 14 after birth. Group A (n = 26), B (n = 22), C (n = 27), and D (n = 18) mice were the same mice as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027102#pone-0027102-g003" target="_blank">figure 3</a>. The values shown are the mean ± S.E. concentrations (ng/ml). The mean values of IgG1/IgG2a ratio were also shown. * <i>p</i><0.05 and **<i>p</i><0.01 are for comparisons with offspring in Group D for PspA-specific IgG subclasses or with offspring on day 0 for the IgG1/IgG2a ratio by ANOVA test with Dunn's multiple comparison test.</p

Protection against fatal systemic pneumococcal infections by maternal immunization with PspA among offspring.

<p>Offspring at 10-days of age were intraperitonally challenged with 1×10⁴ CFU TIGR4 strain (100 µl/mouse) with anesthesia. After challenge, offspring were monitored for 5 days to determine survival. Group A (n = 24), B (n = 28), C (n = 24), and D (n = 38) mice were produced in the same manner as the corresponding groups in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027102#pone-0027102-g003" target="_blank">figure 3</a>. Group A mice are offspring delivered from PspA-immunized mothers and breast-fed by the same mothers (n = 24). Group B mice are offspring from sham-immunized mothers and breast-fed by PspA-immunized mothers (n = 28). Group C mice are offspring from PspA-immunized mothers and breast-fed by sham-immunized mothers (n = 24). Group D mice are offspring from sham-immunized mothers and breast-fed by the same mothers (n = 38). * <i>p</i><0.01 when compared with control offspring in Group D by Kaplan-Meier test with Log rank test.</p

Anti-PspA specific antibodies in sera and milk of mother mice.

<p>Female mice were intranasally immunized twice each week with 1 µg of rPspA and 4 µg CTB for first 2 weeks and with 1 µg rPspA alone for the last week. The levels of anti-PspA specific IgG, IgA and IgM antibodies in sera (A) and breast milk (B) were determined by PspA-specific ELISA on day 0, 7 and 14 after the birth. The values shown are the mean ± S.E. concentrations (ng/ml) taken from PspA-immunized mother (n = 16) and sham-immunized mother (n = 14). The levels of anti-PspA specific antibodies in sera and breast milk from sham-immunized mice were below the limit of detection.</p

Cross-protection against fatal infections with pneumococcal strains expressing family 1 PspA.

<p>As in the prior studies the mother mice were immunized with a rPspA2 of strain TIGR4. Offspring at 10-days of age were intraperitoneally challenged with D39 (PspA1, serotype 2; 50 CFUs/mouse), BG7322 (PspA1, serotype 6B; 20 CFUs/mouse), EF3030 (PspA1, serotype 19F; 5×10⁶ CFUs/mouse), and L82016 (PspA1 serotype 6B, 5×10⁶ CFUs/mouse) in 100 µl with anesthesia. After challenge, the mice were monitored for 10 days to determine the day of death. Group A mice were offspring delivered from PspA-immunized mothers and breast-fed by the same mothers (n = 6 for D39, n = 10 for BG7322, n = 7 for EF3030, n = 6 for L82016). Group B mice were offspring from sham-immunized mothers and breast-fed by a PspA-immunized mothers (n = 7 for D39, n = 11 for BG7322, n = 4 for EF3030, n = 10 for L82016). Group C were offspring from PspA-immunized mothers and breast-fed by sham-immunized mothers (n = 9 for D39, n = 11 for BG7322, n = 5 for EF3030, n = 11 for L82016). Group D mice were offspring from sham-immunized mothers and breast-fed by the same mothers (n = 9 for D39, n = 5 for BG7322, n = 6 for EF3030, n = 6 for L82016). <i>p</i><0.05 and <i>p</i><0.01 for the indicated comparisons with offspring in Group D by Kruskal-Wallis test with Dunn's multiple comparison test are shown.</p

Single Cell Bottlenecks in the Pathogenesis of <i>Streptococcus pneumoniae</i>

<div><p>Herein, we studied a virulent isolate of the leading bacterial pathogen <i>Streptococcus pneumoniae</i> in an infant mouse model of colonization, disease and transmission, both with and without influenza A (IAV) co-infection. To identify vulnerable points in the multiple steps involved in pneumococcal pathogenesis, this model was utilized for a comprehensive analysis of population bottlenecks. Our findings reveal that in the setting of IAV co-infection the organism must pass through single cell bottlenecks during bloodstream invasion from the nasopharynx within the host and in transmission between hosts. Passage through these bottlenecks was not associated with genetic adaptation by the pathogen. The bottleneck in transmission occurred between bacterial exit from one host and establishment in another explaining why the number of shed organisms in secretions is critical to overcoming it. These observations demonstrate how viral infection, and TLR-dependent innate immune responses it stimulates and that are required to control it, drive bacterial contagion.</p></div

Increased pneumococcal shedding among <i>tlr2</i>^<i>-/-</i> mice or by daily treatment with Toll-like receptor (TLR) agonists.

<p><b>A.</b> Wildtype (black circle) or <i>tlr2</i>^<i>-/-</i> pups (black triangle) were infected with strain P1547 on day 4, and IAV on day 8 of age as indicated. Daily nasal secretions were collected between day 8–12 of age. Each dot represents a single mouse. Statistical differences between the two groups in each day were evaluated by using Mann-Whitney U test. * <i>p</i><0.05 and **<i>p</i><0.01. <b>B, C, D.</b> Wildtype pups were infected with strain P1547 on day 4, and given a daily IN dose of PBS, Pam3Cys, poly-ICLC or LPS between days 8 and 12. Statistical analyses between four groups were performed by Kruskal-Wallis test (Dunn’s multiple comparison test). **<i>p</i><0.01, *** <i>p</i><0.001, **** <i>p</i><0.0001 and n.s. not significant. <b>B.</b> Shedding (combined values from days 9 through12) was compared by quantitative culture of secretions. <b>C.</b> Colonization was compared by quantitative culture of nasal lavages obtained at day 12 of age. <b>D.</b> The number of neutrophils (PMNs) in the nasal lavages were counted by flow cytometry by gating on CD11b⁺, Ly6G⁺ events.</p

Analysis of tight population bottlenecks.

<p><b>A.</b> A tight population bottleneck exists in bacteremia after IN challenge. Pups were inoculated with three mutant mixture on day 4 and IAV (above) or PBS (below) on day 8 of age. Data with IAV is from three representative experiments. Each vertical tick mark on the x-axis represents results of blood cultures obtained when the pups showed signs of sepsis from one pup. <b>B.</b> Most transmission events originate from a single organism. Two representative litters out of five are shown. One pup was inoculated with each of three marked mutants on day 4 of age (index mice) and returned to the littermates (contacts). <b>C.</b> Multiple entry events may occur during transmission. Three pups were each inoculated with one of the three marked mutants on day 4 of age (index mice) and returned to the littermates (contacts). <b>B, C.</b> On day 8, all pups were infected with IAV. The bacterial density in the nasal lavage of each mouse on day of age 12 is shown. Each vertical tick mark on the x-axis represents results of cultures nasal lavages from a single pup.</p